Use of the irritating principal oleocanthal in olive oil, as well as structurally and functionally similar compounds

ABSTRACT

The invention provides methods of synthesizing the purified enantiomers of oleocanthal. The invention further provides methods of using oleocanthals in various formulations including, food additives; pharmaceuticals; cosmetics; animal repellants; and discovery tools for mammalian irritation receptor genes, gene products, alleles, splice variants, alternate transcripts and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/679,136, filed May 9, 2005 and U.S. Provisional Application No.60/703,565, filed Jul. 29, 2005, the disclosures of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to the active principal in olive oil, termedoleocanthal, and methods of using oleocanthals in various formulationsincluding, food additives, pharmaceuticals, cosmetics, animalrepellants, and discovery tools for mammalian irritation receptor genes,gene products, alleles, splice variants, alternate transcripts and thelike.

BACKGROUND OF THE INVENTION

Over forty years ago, Fisher and Griffin suggested that the human oralcavity could be regarded as a pharmacological preparation in situ. Theyproposed that the perceived bitterness intensity of a compound reflectsthe compound's pharmacological activity and potency. As support for thisidea, they pointed out that for several drugs, the active isomer wasmore bitter than the inactive one. There is also a rough correlationbetween the bitter potency of selected toxins and their LD₅₀ values.

In addition to the quality and intensity of a sensation, the perceivedlocation may have pharmacological implications. Many compounds when putin the oral cavity elicit irritation (e.g., burning, stinging, cooling)and, just as for bitter taste, the irritation may serve as a signal ofpotential danger.

Some compounds with site-specific irritation have a beneficial effect. Adesirable attribute of many premium olive oils is the distinctiveirritation or pungency that is unusual because it is almost exclusivelyperceived on the pharynx and not in the mouth.

In 1993, Montedoro and co-workers reported the isolation of a new classof phenolic compounds (1-4), including the dialdehydic and aldehydicforms of ligstroside (5) and oleuropeine (6) from virgin olive oils(Montedoro, G. et al. (1993) J. Agric. Food Chem. 41:2228-2234) (SeeFIG. 1 for structures). These phenolic compounds comprise importantminor constituents of virgin olive oils that have been implicated in theorganoleptic characteristics including bitterness, pungency, andastringency (Andrewes, P. et al. (2003) J. Agric. Food Chem.57:1415-1420). In addition, these agents have been suggested tocontribute to the oxidative stability of virgin olive oil and as suchare associated with health benefits of olive oils, specifically theirantioxidant/anticancer activities (Owen, R. W. et al. (2000) Food Chem.Toxicology 38:647-659; Owen, R. W. et al. (2000) Eur. J. Cancer 36(10):1235-1247; Baldioli, M. et al. (1996) J. Am. Oil Chem. Soc. 73(11):1589-1593; Manna, C. et al (2002) J. Agric. Food Chem. 50(22):6521-6526). Similar structural features have been reported in theconstituents of the Jasminum (Somanadhan, B. et al. (1998) Planta Medica64:246-50; Takenaka, Y. et al. (2002) Chem. & Pharm. Bull 50(3):384-389) and related plant species (Takenaka, Y. et al. (2002)Phytochemistry 59 (7):779-787). It has been shown that both ibuprofenand a Mediterranean diet (i.e., high in olive oil) both decrease therisk/incidence for breast and lung cancer.

In 2003, Busch and co-workers at Unilever Research and DevelopmentVlaardingen (The Netherlands) identified deacetoxydialdehydicligstroside aglycone as a principal contributor to the potent pungent(burning) sensation at the back of throat associated with high qualityvirgin olive oils (Andrewes, P. et al. (2003) J. Agric. Food Chem.57:1415-1420). Studies at Firmenich, Inc., reached the same conclusion(Firmenich, Inc. study). The structure of 1 was assigned,

employing a series of 1 and 2D NMR experiments (Andrewes, P. et al.(2003) J. Agric. Food Chem. 57:1415-1420), in conjunction withcomparison to literature data (Montedoro, G. et al. (1993) J. Agric.Food Chem. 41:2228-2234). The absolute stereochemistry remainedundetermined. That 1 was responsible for the strong pungent (burning)sensation at the back of the throat was based on an extensive series ofHPLC fraction analysis, omission analysis and correlation, andhydrolysis studies, in conjunction with human sensory studies. Andreweset al., however, acknowledged that “a coelution compound causing theburning sensation” could not be eliminated without completing asynthesis of 1, which they stated to be “extremely challenging.”

SUMMARY OF THE INVENTION

The invention provides the enantioselective total syntheses of bothenantiomers of oleocanthal 1 (FIG. 1), which not only confirms thestructure, but also permits the assignment of absolute stereochemistryof the olive oil irritant. The synthesis provides an effective route toboth enantiomers for further biological/sensory evaluation. Studiesdemonstrate that the levorotary (−)-enantiomer of 1 (FIG. 1) isresponsible for the organoleptic properties experienced with premiumolive oils at back of the throat.

The invention therefore provides isolated and purifieddeacetoxydialdehydic ligstroside aglycone, which we term oleocanthal.The invention also provides functional derivatives of oleocanthal havingthe general formula:

wherein:

R₁ and R₄ are independently H or OR₅

R₂ and R₃ are independently CHO, or COOR₅

R₅ is a H, C₁-C₅ alkyl, or a glycoside

X is O, NH or CH₂

Y is C═CHCH₃, or CH—COOR₅

Z is C═O or CH—OR₅

A is CH₂, or CH—COOR₅

The compounds of Formula I, including oleocanthal, are collectivelyreferred to herein as “oleocanthals.” The Term “oleocanthal”specifically refers to deacetoxydialdehydic ligstroside aglycone.

The invention provides methods of synthesizing the purified enantiomersof oleocanthal.

The invention further provides methods of using oleocanthals in variousformulations including, food additives (e.g., flavor enhancers,sweetness inhibitors, spices, flavorings, and preservatives);pharmaceuticals (e.g., antioxidants, micro-G protein and associatedkinase inhibitors, Aβ42 inhibitors, presenilin modifiers, γ-secretaseinhibitors, non-steroidal anti-inflammatories, anti-pyretics, cold andflu symptom relievers, COX-1, Cox-2 inhibitors, Cox-3 inhibitors,lipoxygenase inhibitors, and wound healers); cosmetics; animalrepellants; and discovery tools for mammalian irritation receptor genes,gene products, alleles, splice variants, alternate transcripts and thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows phenolic compounds (1-4), including the dialdehydic andaldehydic forms of ligstroside (5) and oleuropeine (6).

FIG. 2 shows a graph of the irritation intensity of various olive oilsplotted against their concentrations of oleocanthal.

FIG. 3 shows the synthetic scheme of (−)-oleocanthal.

FIG. 4 shows the synthetic scheme of (+)-oleocanthal.

FIG. 5 shows the scheme of a Structure Activity Relationship (SAR)Study.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The reference works, patents, patent applications, and scientificliterature that are referred to herein establish the knowledge of thosewith skill in the art and are hereby incorporated by reference in theirentirety to the same extent as if each was specifically and individuallyindicated to be incorporated by reference. Any conflict between anyreference cited herein and the specific teachings of this specificationshall be resolved in favor of the latter.

Various definitions are made throughout this document. Most words havethe meaning that would be attributed to those words by one skilled inthe art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of the presentinvention as a whole and as are typically understood by those skilled inthe art. Any conflict between an art-understood definition of a word orphrase and a definition of the word or phrase as specifically taught inthis specification shall be resolved in favor of the latter. Headingsused herein are for convenience and are not to be construed as limiting.

Standard reference works setting forth the general principles ofchemical synthesis are well known to those of skill in the art andinclude, for example, A. I. Vogel, VOGEL'S TEXTBOOK OF PRACTICAL ORGANICCHEMISTRY (5^(TH) EDITION) WILEY, N.Y. 1989; and ORGANIC SYNTHESES. 9collective volumes; Index for vol. 1-8; Wiley, N.Y.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, 1998; Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y.,1989; Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton, 1995; McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford, 1991.

As used herein, “taste perception” refers to a response (e.g.,biochemical, behavioral) or sensitivity to a taste stimulus. “Tastestimulus” as used herein refers to any compound that elicits, forexample at the biochemical level (e.g., activation or inhibition of ataste receptor) or behavioral level (e.g., preference, indifference, ordistaste), a taste response which would be perceived by a mammal as atleast one of the five taste elements, including sweet, salty, sour,bitter, and umami. “Taste perception” or “taste stimulus,” or variantsthereof, does not require, though it does include, transmission of aneural signal resulting in in vivo sensation of taste by a mammal.Modification of taste perception includes an alteration of (enhancementof, reduction to, or change to) a biochemical response, an ingestiveresponse, a taste preference, or general behavior of a mammal inresponse to a compound.

“Acyl” refers to a straight or branched alkyl-C═O group. “Thioacyl”refers to a straight or branched alkyl-C═S group. Preferred acyl andthioacyl groups are lower alkanoyl and lower thioalkanoyl having from 1to about 6 carbon atoms in the alkyl group, and all combinations andsubcombinations of ranges therein.

“Alkyl” refers to a saturated aliphatic hydrocarbon group which may bestraight or branched and having from 1 to about 20 carbon atoms in thechain, and all combinations and subcombinations of ranges therein.Preferred alkyl groups may be straight or branched and have from 1 toabout 10 carbon atoms in the chain. Branched means that a lower alkylgroup such as, for example, methyl, ethyl or propyl, is attached to alinear alkyl chain.

“Lower alkyl” refers to an alkyl group having from 1 to about 6 carbons,and all combinations and subcombinations of ranges therein.

“Cycloalkyl” refers to an aliphatic ring having from about 3 to about 10carbon atoms in the ring, and all combinations and subcombinations ofranges therein. Preferred cycloalkyl groups have from about 4 to about 7carbon atoms in the ring.

“Carbamoyl” refers to an H₂N—C═O group. Alkylcarbamoyl anddialkylcarbamoyl means that the nitrogen of the carbamoyl is substitutedby one or two alkyl groups, respectively.

“Carboxyl” refers to a COOH group.

“Alkoxy” refers to an alkyl-O group in which “alkyl” is as previouslydescribed. Lower alkoxy groups are preferred. Exemplary alkoxy groupsinclude, for example, methoxy, ethoxy, n-propoxy, i-propoxy andn-butoxy.

“Alkoxyalkyl” refers to an alkyl group, as previously described,substituted by an alkoxy group, as previously described.

“Alkoxycarbonyl” refers to an alkoxy-C═O group.

“Aryl” refers to an aromatic carbocyclic radical containing from about 6to about 10 carbons, and all combinations and subcombinations of rangestherein. Exemplary aryl groups include phenyl and naphthyl.

“Aralkyl” means an alkyl group substituted by an aryl radical.“Optionally substituted aralkyl” and “optionally substituted aryl” meansthat the aryl group, or the aryl group of the aralkyl group, may besubstituted with one or more substituents which include, for example,alkyl, alkoxy, amino, nitro, carboxy, carboalkoxy, cyano, alkyl amino,halo, hydroxy, hydroxyalkyl, mercaptyl, alkylmercaptyl, trihaloalkyl,carboxyalkyl or carbamoyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—C═O group.

“Aryloxycarbonyl” refers to an aryl-O—C═O group.

“Carbalkoxy” refers to a carboxyl substituent esterified with an alcoholof the formula C_(n)H_(2n+1)OH, wherein n is from 1 to about 6.

“Halogen” (or “halo”) refers to chlorine (chloro), fluorine (fluoro),bromine (bromo) or iodine (iodo). Preferred among the halogens (orhalos) is chlorine (or chloro).

“Heterocyclyl” refers to a ring structure containing from about 4 toabout 10 members in which one or more of the atoms in the ring is anelement other than carbon, e.g., N, O or S. Heterocyclyl groups may bearomatic or non-aromatic, i.e., the rings may be saturated, partiallyunsaturated, or fully unsaturated. Preferred heterocyclyl groupsinclude, for example, pyridyl, pyridazinyl, pyrimidinyl, isoquinolinyl,quinolinyl, quinazolinyl, imidazolyl, pyrrolyl, furanyl, thienyl,thiazolyl, benzothiazolyl, piperidinyl, pyrrolidinyl, tetrahydrofuranyl,tetrahydropyranyl, and morphonlinyl groups.

“Optionally substituted heterocyclyl” means that the heterocyclyl groupmay be substituted by one or more substituents wherein the substituentsinclude, for example, alkoxy, alkylamino, aryl, carbalkoxy, carbamoyl,cyano, halo, heterocyclyl, trihalomethyl, hydroxy, mercaptyl,alkylmercaptyl and nitro.

“Hydroxyalkyl” refers to an alkyl group substituted by a hydroxy group.Hydroxy lower alkyl groups are preferred. Exemplary preferred groupsinclude, for example, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl and3-hydroxypropyl.

“Hydrogenation catalyst” refers to any compounds known in the art oforganic synthesis to facilitate the addition of hydrogen. Hydrogenationcatalysts include, but are not limited to palladium on carbon, palladiumhydroxide on carbon, palladium on calcium carbonate poisoned with lead,and platinum on carbon.

“Sulfonating agent” refers to any reagents known in the art of organicsynthesis to react with an alcohol to provide a sulfonate ester.Examples include, but are not limited to methanesulfonyl chloride,methanesulfonic anhydride, trifluoromethane sulfonyl chloride,trifluoromethane sulfonic anhydride, benzene sulfonyl chloride,p-toluenesulfonyl chloride, a p-toluenesulfonyl anhydride. “Sulfonateester” includes groups which result when a sulfonating agent is reactedwith an alcohol in the presence of an acid scavenger to give a compoundof form —OA, wherein A is SO₂R′, with R′ deriving from the sulfonatingagent.

“Reducing agent” refers to any reagents known in the art of organicsynthesis to reduce the oxidation state of a carbon atom, for example,by reducing a ketone to an alcohol. Reducing agents include, but are notlimited to hydride derivatives, such as borohydrides, including lithiumborohydride and sodium borohydrides.

“Methylating agent” refers to any reagent known in the art of organicsynthesis to donate a methyl group to an alcohol to form an ether.Methylating agents include, but are not limited to methylhalides such asmethyliodide, methylchloride, methylbromide, and dimethylsulfate.

“Acid scavenger” refers to any species known in the art of organicsynthesis capable of accepting a proton without reacting with thestarting material or product.

“Concatenated” refers to multi-step processes (i.e., processescontaining two or more steps) wherein the steps may be performed in asubstantially continuous or sequential manner, preferably without thenecessity for interim isolation and/or purification of the intermediatecompounds.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. Thus, the term “acidaddition salt” refers to the corresponding salt derivative of a parentcompound which has been prepared by the addition of an acid. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. Certain acidic or basic compounds may exist aszwitterions. All forms of the compounds, including free acid, free baseand zwitterions, are contemplated to be within the scope of the presentinvention.

The reactions of the synthetic methods described and claimed herein maybe carried out in suitable solvents which may be readily selected by oneskilled in the art of organic synthesis. Generally, suitable solventsare solvents which are substantially non-reactive with the startingmaterials (reactants), the intermediates, or products at thetemperatures at which the reactions are carried out, i.e., temperatureswhich may range from the solvent's freezing temperature to the solvent'sboiling temperature. A given reaction may be carried out in one solventor a mixture of more than one solvent. Depending on the particularreaction, suitable solvents for a particular work-up following thereaction may be selected. Suitable solvents, as used herein may include,by way of example and without limitation, chlorinated solvents,hydrocarbon solvents, aromatic solvents, ether solvents, proticsolvents, polar aprotic solvents, and mixtures thereof.

Suitable halogenated solvents include, but are not limited to carbontetrachloride, bromodichloromethane, dibromochloromethane, bromoform,chloroform, bromochloromethane, dibromomethane, butyl chloride,dichloromethane, tetrachloroethylene, trichloroethylene,1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethane,2-chloropropane, hexafluorobenzene, 1,2,4-trichlorobenzene,o-dichlorobenzene, chlorobenzene, fluorobenzene, fluorotrichloromethane,chlorotrifluoromethane, bromotrifluoromethane, carbon tetrafluoride,dichlorofluoromethane, chlorodifluoromethane, trifluoromethane,1,2-dichlorotetrafluorethane and hexafluoroethane.

Suitable hydrocarbon solvents include, but are not limited to alkane oraromatic solvents such as cyclohexane, pentane, hexane, toluene,cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o-, orp-xylene, octane, indane, nonane, benzene, ethylbenzene, and m-, o-, orp-xylene.

Suitable ether solvents include, but are not limited todimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan,diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethylether, diethylene glycol dimethyl ether, diethylene glycol diethylether, triethylene glycol diisopropyl ether, anisole, or t-butyl methylether.

Suitable protic solvents include, but are not limited to water,methanol, ethanol, 2-nitroethanol, 2-fluoroethanol,2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol,2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butylalcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, or 3-pentanol,neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethylether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol,phenol, and glycerol.

Suitable aprotic solvents include, but are not limited todimethylformamide (DMF), dimethylacetamide (DMAC),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP),formamide, N-methylacetamide, N-methylformamide, acetonitrile (ACN),dimethylsulfoxide (DMSO), propionitrile, ethyl formate, methyl acetate,hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate,isopropyl acetate, t-butyl acetate, sulfolane, N,N-dimethylpropionamide,nitromethane, nitrobenzene, and hexamethylphosphoramide.

The term “substantially pure form,” as used herein, means that thecompounds prepared using the present processes may preferably besubstantially devoid of organic impurities. The term “organicimpurities,” as used herein, refers to organic materials, compounds,etc., other than the desired product, that may be typically associatedwith synthetic organic chemical transformations including, for example,unreacted starting reagents, unreacted intermediate compounds, and thelike. In preferred form, the present processes may provide compoundsthat are at least about 75% pure, as measured by standard analyticaltechniques such as, for example, HPLC. Preferably, the compoundsprepared using the present processes may be at least about 80% pure,with a purity of at least about 85% being more preferred. Even morepreferably, the compounds prepared using the present processes may be atleast about 90% pure, with a purity of at least about 95% being morepreferred. In particularly preferred embodiments, the compounds preparedusing the present processes may be more than about 95% pure, with apurity of about 100% being especially preferred.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen. Exemplary substituents include, forexample, halo (e.g., F, Cl, Br, I), alkyl, alkenyl, alkynyl, aralkyl,aryl, heteroaryl, heterocyclyl, hydroxyl (OH), nitro (NO₂), nitrosyl(NO), cyano (CN), cyanato (CNO), thiocyanato (SCN), amino (e.g., NH₂,NHR′, NR′₂), azido (N₃), carboxyl (COOH), C(O)R′, OR′, C(O)OR′,NHC(O)R′, aminocarbonyl, thiol, thiolato (SR′), sulfonic acid (SO₃H),phosphonic acid (PO₃H), SO₂R′, phosphino (PH₂, PHR′, PR′₂), silyl(SiR′₃, SiHR′₂, SiH₂R′, SiH₃) and the like. In relation to theaforementioned substituents, each moiety R′ can be, independently, anyof H, alkyl, aryl, aralkyl, heteroaryl, or heterocyclyl, for example.

Processes of the present invention may yield mixtures of diastereomers.Thus, in some embodiments, processes may, if desired, include aseparation step to isolate diastereomers. Methods for separation ofdiastereomers are well known in the art and include, for example, chiralcolumn chromatography, HPLC, re-crystallization, or classical resolutionmethods involving selective reactivity. In some embodiments, asymmetricsynthesis may be used to produce a specific diastereomer.

As used herein “polynucleotide” refers to a nucleic acid molecule andincludes genomic DNA, cDNA, RNA, mRNA, mixed polymers, recombinantnucleic acids, fragments and variants thereof, and the like.Polynucleotide fragments of the invention comprise at least 10, andpreferably at least 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, or100 consecutive nucleotides of a reference polynucleotide. Thepolynucleotides include sense and antisense strands. The polynucleotidesmay be naturally occurring or non-naturally occurring polynucleotides. A“synthesized polynucleotide” as used herein refers to polynucleotidesproduced by purely chemical, as opposed to enzymatic, methods. “Wholly”synthesized DNA sequences are therefore produced entirely by chemicalmeans, and “partially” synthesized DNAs embrace those wherein onlyportions of the resulting DNA were produced by chemical means. Thepolynucleotides of the invention may be single- or double-stranded. Thepolynucleotides of the invention may be chemically modified and maycontain non-natural or derivatized nucleotide bases as will be readilyappreciated by those skilled in the art. Such modifications include, forexample, labels, methylation, substitution of one or more nucleotideswith an analog, internucleotide modifications such as uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.), charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), pendent moieties (e.g., polypeptides, etc.),intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators,and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Alsoincluded are synthetic molecules that mimic polynucleotides in theirability to bind to a designated sequence via hydrogen bonding and otherchemical interactions. Such molecules are known in the art and include,for example, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

“Recombinant nucleic acid” is a nucleic acid generated by combination oftwo segments of nucleotide sequence. The combination may be, forexample, by chemical means or by genetic engineering.

As used herein, “polynucleotide amplification” refers to a broad rangeof techniques for increasing the number of copies of specificpolynucleotide sequences. Typically, amplification of either or bothstrand(s) of the target nucleic acid comprises the use of one or morenucleic acid-modifying enzymes, such as a DNA polymerase, ligase, RNApolymerase, or RNA-dependent reverse transcriptase. Examples ofpolynucleotide amplification include, but are not limited to, polymerasechain reaction (PCR), nucleic acid sequence based amplification (NASB),self-sustained sequence replication (3SR), strand displacementactivation (SDA), ligase chain reaction, Qβ replicase system, and thelike. A wide variety of alternative cloning and in vitro amplificationmethodologies are well known to those skilled in the art. Examples ofthese techniques are found in, for example, Berger et al., Guide toMolecular Cloning Techniques, METHODS IN ENZYMOLOGY 152, Academic Press,Inc., San Diego, Calif. (Berger), which is incorporated herein byreference in its entirety.

As used herein, the term “oligonucleotide” or “primer” refers to aseries of linked nucleotide residues which has a sufficient number ofbases to be used in a polymerase chain reaction (PCR). This shortsequence is based on (or designed from) a genomic or cDNA sequence andis used to amplify, confirm, or reveal the presence of an identical,similar, or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence having atleast about 10 nucleotides and as many as about 50 nucleotides, oftenabout 12 or 15 to about 30 nucleotides. They are chemically synthesizedand may be used as probes. “Primer pair” refers to a set of primersincluding a 5′ upstream primer that hybridizes with the 5′ end of atarget sequence to be amplified and a 3′ downstream primer thathybridizes with the complement of the 3′ end of the target sequence tobe amplified.

As used herein, the term “probe” refers to nucleic acid sequences ofvariable length, for example between at least about 10 and as many asabout 8,500 nucleotides, depending on use. Probes are used in thedetection of identical, similar, or complementary target nucleic acidsequences, which target sequences may be single- or double-stranded.Longer probes are usually obtained from a natural or recombinant source,are highly specific, and are much slower to hybridize than oligomers, orshorter probes. They may be single- or double-stranded and are carefullydesigned to have specificity in PCR, hybridization membrane-based, orELISA-like technologies.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a probe, primer,or oligonucleotide will hybridize to its target sequence, but to aminimal number of or no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences will hybridize with specificity to their propercomplements at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present in excess, at T_(m), 50% of theprobes are hybridized to their complements at equilibrium. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and may be in excess of 45° C.Stringent salt conditions will ordinarily be less than 1.0 M, typicallyless than 0.5 M, and may be less than 0.2 M. Typically, stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes, primers, or oligonucleotides (e.g., 10 to 50nucleotides) and at least about 60° C. for longer probes, primers, oroligonucleotides. Stringent conditions may also be achieved with theaddition of destabilizing agents, such as formamide.

As used herein “antisense oligonucleotide” refers to a nucleic acidmolecule that is complementary to at least a portion of a targetnucleotide sequence of interest and specifically hybridizes to thetarget nucleotide sequence under physiological conditions. The term“double stranded RNA” or “dsRNA” as used herein refers to adouble-stranded RNA molecule capable of RNA interference, includingsmall interfering RNA (siRNA) (see for example, Bass (2001) Nature411:428-429; Elbashir et al. (2001) Nature, 411:494-498).

As used herein, the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.

The term “marker gene” or “reporter gene” refers to a gene encoding aproduct that, when expressed, confers a phenotype at the physical,morphologic, or biochemical level on a transformed cell that is easilyidentifiable, either directly or indirectly, by standard techniques andincludes, but is not limited to, genes encoding proteins that conferresistance to toxins or antibiotics such as ampicillin, neomycin, andmethotrexate; genes encoding proteins that complement auxotrophicdeficiencies; and genes encoding proteins that supply criticalcomponents not available from complex media. Examples of marker genesinclude green fluorescent protein (GFP), red fluorescent protein(DsRed), alkaline phosphatase (AP), β-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferase (NEOr, G418r) dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ(encoding β-galactosidase), β-lactamase, luciferase (luc), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional sequences that can serve thefunction of a marker or reporter. Thus, this list is merely meant toshow examples of what can be used and is not meant to limit theinvention.

As used herein, the term “promoter” refers to a regulatory element thatregulates, controls, or drives expression of a nucleic acid molecule ofinterest and can be derived from sources such as from adenovirus, SV40,parvoviruses, vaccinia virus, cytomegalovirus, or mammalian genomic DNA.Examples of suitable promoters include, but are not limited to, CMV,MSH2, trp, lac, phage, and TRNA promoters. Suitable promoters that canbe used in yeast include, but are not limited to, such constitutivepromoters as 3-phosphoglycerate kinase and various other glycolyticenzyme gene promoters such as enolase or glyceraldehydes-3-phosphatedehydrogenase, or such inducible promoters as the alcohol dehydrogenase2 promoter or metallothionine promoter. Again, as with many of thestandard procedures associated with the practice of the invention,skilled artisans will be aware of additional promoters that can servethe function of directing the expression of a marker or reporter. Thus,the list is merely meant to show examples of what can be used and is notmeant to limit the invention.

“Operably linked” refers to juxtaposition wherein the components are ina functional relationship. For example, a promoter is operably linked orconnected to a coding sequence if it controls the transcription orexpression of the sequence.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein. “Polypeptide” refers to a polymer of amino acidswithout referring to a specific length. Polypeptides of the inventioninclude peptide fragments, derivatives, and fusion proteins. Peptidefragments preferably have at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, or 100 amino acids. Some peptide fragments of theinvention are biologically active. Biological activities includeimmunogenicity, ligand binding, and activity associated with thereference peptide. Immunogenic peptides and fragments of the inventiongenerate an epitope-specific immune response, wherein “epitope” refersto an immunogenic determinant of a peptide and preferably contains atleast three, five, eight, nine, ten, fifteen, twenty, thirty, forty,forty-five, or fifty amino acids. Some immunogenic peptides of theinvention generate an immune response specific to that peptide.Polypeptides of the invention include naturally occurring andnon-naturally occurring peptides. The term includes modifiedpolypeptides (wherein examples of such modifications includeglycosylation, acetylation, phosphorylation, carboxylation,ubiquitination, labeling, etc.), analogs (such as non-naturallyoccurring amino acids, substituted linkages, etc.), and functionalmimetics. A variety of methods for labeling polypeptides are well knownin the art and include radioactive isotopes such as ³²P or ³⁵S, ligandsthat bind to labeled antiligands (e.g., antibodies), fluorophores,chemiluminescent agents, enzymes, and antiligands.

As used herein, the term “amino acid” denotes a molecule containing bothan amino group and a carboxyl group. In some embodiments, the aminoacids are α-, β, γ- or δ-amino acids, including their stereoisomers andracemates. As used herein the term “L-amino acid” denotes an α-aminoacid having the L configuration around the α-carbon, that is, acarboxylic acid of general formula CH(COOH)(NH₂)-(side chain), havingthe L-configuration. The term “D-amino acid” similarly denotes acarboxylic acid of general formula CH(COOH)(NH₂)-(side chain), havingthe D-configuration around the α-carbon. Side chains of L-amino acidsinclude naturally occurring and non-naturally occurring moieties.Non-naturally occurring (i.e., unnatural) amino acid side chains aremoieties that are used in place of naturally occurring amino acid sidechains in, for example, amino acid analogs. Amino acid substituents maybe attached, for example, through their carbonyl groups through theoxygen or carbonyl carbon thereof, or through their amino groups, orthrough functionalities residing on their side chain portions.

The amino acid sequences are presented in the amino (N) to carboxy (C)direction, from left to right. The N-terminal α-amino group and theC-terminal β-carboxy groups are not depicted in the sequence. Thenucleotide sequences are presented by single strands only, in the 5′ to3′ direction, from left to right. Nucleotides and amino acids arerepresented in the manner recommended by the IUPAC-IUB BiochemicalNomenclature Commission, or amino acids are represented by their threeletters code designations.

As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof. Binding includes ionic, non-ionic,Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Thephysical interaction, the binding, can be either direct or indirect,indirect being through or due to the effects of another protein orcompound. Direct binding refers to interactions that do not take placethrough or due to the effect of another protein or compound but insteadare without other substantial chemical intermediates. Binding may bedetected in many different manners. As a non-limiting example, thephysical binding interaction between two molecules can be detected usinga labeled compound. Other methods of detecting binding are well-known tothose of skill in the art.

As used herein, the term “contacting” means bringing together, eitherdirectly or indirectly, a compound into physical proximity to a moleculeof interest. Contacting may occur, for example, in any number ofbuffers, salts, solutions, or in a cell or cell extract.

As used herein, the terms “modulates” or “modifies” means an increase ordecrease in the amount, quality, or effect of a particular activity orprotein. “Modulators” refer to any inhibitory or activating moleculesidentified using in vitro and in vivo assays for, e.g., agonists,antagonists, and their homologues, including fragments, variants, andmimetics, as defined herein, that exert substantially the samebiological activity as the molecule. “Inhibitors” or “antagonists” aremodulating compounds that reduce, decrease, block, prevent, delayactivation, inactivate, desensitize, or downregulate the biologicalactivity or expression of a molecule or pathway of interest. “Inducers,”“activators,” or “agonists” are modulating compounds that increase,induce, stimulate, open, activate, facilitate, enhance activation,sensitize, or upregulate a molecule or pathway of interest. In somepreferred embodiments of the invention, the level of inhibition orupregulation of the expression or biological activity of a molecule orpathway of interest refers to a decrease (inhibition or downregulation)or increase (upregulation) of greater than about 50%, 60%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Theinhibition or upregulation may be direct, i.e., operate on the moleculeor pathway of interest itself, or indirect, i.e., operate on a moleculeor pathway that affects the molecule or pathway of interest.

A “purified” or “substantially purified” polynucleotide or polypeptideis substantially separated from other cellular components that naturallyaccompany a native (or wild-type) nucleic acid or polypeptide and/orfrom other impurities (e.g., agarose gel). A purified polypeptide orprotein will comprise about 60% to more than 99% w/w of a sample, andmay be about 90%, about 95%, or about 98% pure. As used herein, the term“isolated” refers to a molecule that has been removed from its nativeenvironment. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules.

“About” as used herein refers to +/−10% of the reference value.

As used herein, “variant” nucleotide or amino acid sequences refer tohomologues, including, for example, isoforms, species variants, allelicvariants, and fragments of the sequence of interest. “Homologousnucleotide sequence” or “homologous amino acid sequence,” or variationsthereof, refers to sequences characterized by a relative identity, atthe nucleotide level with respect to a reference sequence, or homologyat the amino acid level, of at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%,preferably at least about 90%, at least about 95%, at least about 98%,or at least about 99%, and more preferably 100%, to a referencesequence, or portion or fragment thereof encoding or having a functionaldomain.

As is well known in the art, because of the degeneracy of the geneticcode, there are numerous DNA and RNA molecules that can code for thesame polypeptide as that encoded by a nucleotide sequence of interest.The present invention, therefore, contemplates those other DNA and RNAmolecules which, on expression, encode a polypeptide encoded by thenucleic acid molecule of interest. DNA and RNA molecules other thanthose specifically disclosed herein characterized simply by a change ina codon for a particular amino acid, are within the scope of thisinvention.

Amino acid “insertions,” “substitutions” or “deletions” are changes toor within an amino acid sequence. The variation allowed in a particularamino acid sequence may be experimentally determined by producing thepeptide synthetically or by systematically making insertions, deletions,or substitutions of nucleotides in the nucleic acid sequence usingrecombinant DNA techniques. Alterations of the naturally occurring aminoacid sequence can be accomplished by any of a number of knowntechniques. For example, mutations can be introduced into thepolynucleotide encoding a polypeptide at particular locations byprocedures well known to the skilled artisan, such asoligonucleotide-directed mutagenesis.

A chemical variant of the present invention may exhibit substantiallythe biological activity of a naturally occurring oleocanthal, or haveimproved activity. “Biological activity” as used herein refers to thelevel of a particular function (for example, antioxidant activity,anti-inflammatory activity, etc.) of a molecule or pathway of interestin a biological system. “Wild-type biological activity” refers to thenormal level of function of a molecule or pathway of interest. “Reducedbiological activity” refers to a decreased level of function of amolecule or pathway of interest relative to a reference level ofbiological activity of that molecule or pathway. “Increased biologicalactivity” refers to an increased level of function of a molecule orpathway of interest relative to a reference level of biological activityof that molecule or pathway. For example, increased biological activitymay refer to an increased level of biological activity relative to thewild-type biological activity of a molecule or pathway of interest.Reference to exhibiting “substantially the biological activity ofnaturally-occurring oleocanthal” indicates that variants within thescope of the invention can comprise substitutions, meaning that one ormore chemical moieties of oleocanthal are replaced by different chemicalmoieties and such compounds retain the biological activity ofoleocanthal, have substantially the same biological activities ofoleocanthal, or have improved biological activity as compared tonaturally-occurring oleocanthal.

A nucleotide and/or amino acid sequence of a nucleic acid molecule orpolypeptide identified by the screening method of the invention may beused to search a nucleotide and amino acid sequence databank for regionsof similarity using Gapped BLAST (Altschul, et al. (1997) Nucl. AcidsRes. 25:3389). Briefly, the BLAST algorithm, which stands for BasicLocal Alignment Search Tool is suitable for determining sequencesimilarity (Altschul, et al. (1990) J. Mol. Biol. 215:403-410). Softwareor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul, et al. (1990) J. Mol. Biol. 215:403-410).These initial neighborhood word hits act as seeds for initiatingsearches to find HSPs containing them. The word hits are extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Extension for the word hits in eachdirection are halted when: (1) the cumulative alignment score falls offby the quantity X from its maximum achieved value; (2) the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or (3) the end of either sequenceis reached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff,et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) alignments (B)of 50, expectation (E) of 10, M=5, N=4, and a comparison of bothstrands. The BLAST algorithm (Karlin, et al. (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877) and Gapped BLAST perform a statistical analysisof the similarity between two sequences. One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a gene or cDNA if thesmallest sum probability in comparison of the test nucleic acid to thereference nucleic acid is less than about 1, preferably less than about0.1, more preferably less than about 0.01, and most preferably less thanabout 0.001.

The term “mimetic” as used herein refers to a compound that issterically similar to a reference compound. Mimetics are structural andfunctional equivalents to the reference compounds.

The terms “patient” and “subject” are used interchangeably herein andinclude, but are not limited to amphibians, birds, dogs, cats, cattle,horses, buffalo, llama, sheep, goats, pigs, rodents, monkeys, apes, andhumans. “Host cell” includes, for example, prokaryotic cells, such asbacterial cells; eukaryotic cells, such as yeast cells and animal cells,including, but not limited to invertebrate cells (e.g., insect cells andnematode cells), amphibian cells (e.g., frog cells), particularlymammalian cells (e.g., human, rodent, canine, feline, caprine, ovine,bovine, equine, porcine, simian); or plant cells. “Rodents” include, forexample, rats and mice. Mammalian cell lines available as hosts forexpression are known in the art and include many immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), N1E-115 (Liles etal., (1986) J. Biol. Chem. 261:5307-5313), PC 12 human hepatocellularcarcinoma cells (e.g., Hep G2).

The term “treatment” as used herein refers to any indicia of success ofprevention, treatment, or amelioration of a disease or condition.Treatment includes any objective or subjective parameter, such as, butnot limited to, abatement, remission, normalization of receptoractivity, reduction in the number or severity of symptoms or sideeffects, or slowing of the rate of degeneration or decline of thepatient. Treatment also includes a prevention of the onset of symptomsin a patient that may be at increased risk for or is suspected of havinga disease or condition but does not yet experience or exhibit symptomsthereof.

As used herein, the term “compound” means any identifiable chemical ormolecule, including, but not limited to a small molecule, peptide,protein, sugar, nucleotide, or nucleic acid. Such compound can benatural or synthetic.

As used herein, “bitter” refers to a basic taste characterized bysolutions of such compounds as quinine, caffeine, and certain otheralkaloids, that are sensed in humans primarily by taste buds at the backof the tongue, which are perceived as acrid, sharp, pungent, or harsh.

As used herein, “sweet” refers to a basic taste characterized bysolutions of sugars (e.g., sucrose and glucose), alcohols, glycols, somesmall molecules and some amino acids that are sensed in humans primarilyby taste buds on the tip of the tongue, which are perceived as agreeableor pleasing.

As used herein, “sour” refers to a basic taste characterized bysolutions of vinegar and the juices of most unripe fruits and having aacid or sharp, tart, or biting taste.

Oleocanthals have the general formula:

wherein:

R₁ and R₄ are independently H or OR₅

R₂ and R₃ are independently CHO, or COOR₅

R₅ is a H, C₁-C₅ alkyl, or a glycoside

X is O, NH or CH₂

Y is C═CHCH₃, or CH—COOR₅

Z is C═O or CH—OR₅

A is CH₂, or CH—COOR₅

“Oleocanthal” is specifically deacetoxydialdehydic ligstroside aglycone,which exists as a single isomer (enantiomer). The (−)-enantiomer is thenatural product and has the following chemical formula:

The enantiomers of oleocanthal may be synthesized and purified by thefollowing methods:

D-ribose may be converted to Formula I with a strong acid (e.g.,hydrochloric acid) in acetone and methanol to yield Formula Ia. Thecompound of Formula Ia may be treated with a halogenation reagent (e.g.,iodine), phosphine (PPh₃) imidazole followed by metal halogen exchange(e.g., BuLi or Zn) induced ring opening to yield an aldehyde of FormulaIIa. Thereafter the compound of Formula IIa may be contacted with aCH₂═CH—MgBr in a suitable solvent (e.g., tetrahydrofuran) to yield acompound of Formula IIIa which is converted to a compound of Formula IVaby treatment with Grubbs catalyst in a suitable solvent (e.g.,dichloromethane (DCM)) followed by treatment with an oxidizing reagent(e.g., pyridinium chlorochromate (PCC)). The compound of Formula IVa iscontacted with hydrogen, palladium in a suitable solvent (e.g., ethylacetate (EtOAc)) to yield (−)-cyclopentanone (Formula Va). The(−)-cyclopentanone (Formula Va) is treated with lithiumhexamethyldisilazide (LHMDS) followed by hexamethylphosphoramide (HMPA),dimethyl zinc and allyl bromoacetate (e.g., methyl, ethyl, tert-butyl)to yield (−)-(3,4-dimethoxy-2-oxo-cyclopentyl)-acetic acid ester(Formula VIa). The compound of Formula VIa is subjected to a Wittigethylnation using ethyltriphenylphosphine bromide (or iodide) at reducedtemperature, preferably −40° C. or less. The ester is hydrolyzed(Formula VIIIa) and the compound of formula VIIIa is contacted with4-hydroxyphenethyl alcohol in the presence of phosphine, dialkylazodicarboxylate (e.g., diethyl or diisopropyl)(DEAD or DIAD) to giveFormula IXa. The vicinal diol moiety is liberated and oxidative cleavageyields the (−)-oleocanthal (Formula Xa). See also FIG. 3.

D-ribose may be converted to Formula XI with a strong acid (e.g.,hydrochloric acid) in acetone to yield Formula XI. The compound ofFormula XI may be treated with methyltriphenylphosphine bromide (oriodide) followed by oxidative cleavage of the diol to yield a compoundof Formula IIb. Thereafter the compound of Formula IIb may be contactedwith a CH₂═CH—MgBr in a suitable solvent (e.g., tetrahydrofuran) toyield a compound of Formula IIIb which is converted to a compound ofFormula IVb by treatment with Grubbs catalyst in a suitable solvent(e.g., dichloromethane (DCM)) followed by treatment with an oxidizingreagent (e.g., pyridinium chlorochromate) (PCC) or MnO₂). The compoundof Formula IVb is contacted with hydrogen, catalyst in a suitablesolvent (e.g., ethyl acetate (EtOAc)) to yield (+)-cyclopentanone(Formula Vb). The (+)-cyclopentanone (Formula Vb) is treated withlithium hexamethyldisilazide (LHMDS) followed by hexamethylphosphoramide (HMPA), dimethyl zinc and alkyl bromoacetate (eg., methyl,ethyl, tert-butyl) to yield (+)-(3,4-dimethoxy-2-oxo-cyclopentyl)-aceticacid ester (Formula VIb). The compound of Formula VIb is subjected to aWittig ethylnation using ethyltriphenylphosphine bromide (or iodide) atreduced temperature, preferably −40° C. or less. The ester is hydrolyzed(Formula VIIIb) and the compound of formula VIIIb is contacted with4-hydroxyphenethyl alcohol in the presence of phosphine, dialkylazodicarboxylate (e.g., diethyl or diisopropyl) (DEAD or DIAD) to givethe Formula IXb. The vicinal diol moiety is liberated and oxidativecleavage yields the (+)-oleocanthal (Formula Xb). See also FIG. 4.

The invention contemplates mimetics of oleocanthal that have the generalformula shown above. Mimetics or mimics of oleocanthal (stericallysimilar compounds formulated to mimic the key portions of the structure)may be designed for pharmaceutical use. Mimetics may be used in the samemanner as oleocanthal, and hence are functional equivalents. Thegeneration of a structural-functional equivalent may be achieved by thetechniques of modeling and chemical design known to those of skill inthe art. It will be understood that all such sterically similarconstructs fall within the scope of the present invention.

The design of mimetics to a known pharmaceutically active compound is aknown approach to the development of pharmaceuticals based on a “lead”compound. This is desirable where, for example, the active compound isdifficult or expensive to synthesize, or where it is unsuitable for aparticular method of administration, e.g., some peptides may beunsuitable active agents for oral compositions as they tend to bequickly degraded by proteases in the alimentary canal.

There are several steps commonly taken in the design of a mimetic.First, the particular parts of the compound that are critical and/orimportant in determining its organoleptic properties are determined. Inthe case of oleocanthal, this can be done, for example, bysystematically varying the R groups of the general formula and testingfor anti-inflammatory activity, such as, for example, by the assaysdescribed in the Examples.

Once the active region of the compound has been identified, itsstructure is modeled according to its physical properties, e.g.,stereochemistry, bonding, size, and/or charge, using data from a rangeof sources, such as, but not limited to, spectroscopic techniques, X-raydiffraction data, and NMR. Computational analysis, similarity mapping(which models the charge and/or volume of the active region, rather thanthe bonding between atoms), and other techniques known to those of skillin the art can be used in this modeling process. In a variant of thisapproach, the three-dimensional structure of the compound is modeled.

A candidate general formula is selected onto which chemical groups thatmimic the oleocanthal can be grafted. The general formula and thechemical groups grafted onto it can conveniently be selected so that themimetic is easy to synthesize, is pharmacologically acceptable, and doesnot degrade in vivo, while retaining the biological activity ofoleocanthal. Further optimization or modification can then be performedto arrive at one or more final mimetics for in vivo or clinical testing.

Uses of Oleocanthals

A. As a Food Additive:

The oleocanthals of the invention provide the characteristic irritationsensation found in premium olive oils. The oleocanthals may be added tolower grade oils to provide for an oil that tastes like premium extravirgin olive oil. As such, the oleocanthals act as a flavorant or flavorenhancer. The oleocanthals and formulations of the invention may also beadded to other foods to enhance the flavor or the food by providing apleasing irritation sensation of olive oil.

The oleocanthals of the invention may be added to foods and oralpharmaceutical preparations and oral hygiene products such astoothpaste, mouthwash, breath-fresheners, films, candies, lozenges toprovide an irritant for the oral product's sensory-irritationexperience.

Oleocanthals may also provide sweetness inhibition, or allow thestructural design of other sweetness inhibitors. Such sweetnessinhibitors are useful when carbohydrates are added for bulking andaltering food body and texture.

Finally, oleocanthals may be used to add an irritant to food forenhancing the flavor and gastronomic experience in a similar fashion toother spices such as chilis, mustards, onions, Szechwan pepper, andginger, for example.

B. Preservative:

The oleocanthal and formulations of the invention may be added directlyto food items to act as a preservative. The food items may be for humanconsumption or animal consumption. Especially preferred food items forthe method of preservation are items which are customarily stored inoil. In this method a suitable and effective amount of oleocanthal or aformulation thereof is added directly to the food item or oil in whichthe food item is stored.

In another embodiment of the invention, the oleocanthal or formulationthereof is used to coat the food item prior to packaging. Theformulation may be sprayed onto the food item or the food item may bedipped in the formulation. In another embodiment, the oleocanthal orformulation thereof is applied to the inside surface of packagingmaterial that is in contact with the food item to prevent spoilage. Thecoating may be a thin film sprayed onto the inner surface or laminatedonto the inner surface, for example. In another embodiment of theinvention, the packaging material used to store the food item isimpregnated with oleocanthal or a formulation thereof. All of theembodiments for incorporating a preservative into packaging materials orfor incorporating a preservative in food are well-known in the art, andany suitable means may be employed. Without wishing to be bound by anyparticular theory of operation, the preservative formulations andoleocanthals possess anti-bacterial and antifungal properties whichallow them to act as preservatives.

C. Pharmaceutical Formulations

When employed as pharmaceuticals, the oleocanthals of this invention areusually administered in the form of pharmaceutical compositions. Thesecompounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. These compounds are effective as both injectable and oralcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which containother active ingredients in addition to the oleocanthal compound(s) withpharmaceutically acceptable carriers. In making the compositions of thisinvention, the active ingredient is usually mixed with an excipient,diluted by an excipient or enclosed within such a carrier which can bein the form of a capsule, sachet, paper or other container. When theexcipient serves as a diluent, it can be a soled, semi-solid, or liquidmaterial, which acts as a vehicle, carrier or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, 1-10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 0.001 to about 1 g, more usually about 1 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. Preferably, the compound of formula I above is employed atabout 20 weight percent of the pharmaceutical composition or less, morepreferably about 15 weight percent or less, with the balance beingpharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It, willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

Of course, additionally, the compositions of the present invention maybe formulated in sustained release form to provide the rate controlledrelease of any one or more of the components to optimize the therapeuticeffects while minimizing undesirable side effects. Suitable dosage formsfor sustained release include layered tablets containing layers ofvarying disintegration rates or controlled release polymeric matricesimpregnated with the active components and shaped in tablet form orcapsules containing such impregnated or encapsulated porous polymericmatrices.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, olive oil, coconut oil, or peanut oil, aswell as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable carrier materials.Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

(1) Cold Symptom Relief:

The oleocanthals of the invention may be used in a method for treat thesymptoms of the cold or flu. Formulations may be prepared containingoleocanthals as the active ingredient, or in combination with otheractive ingredients to be taken orally, rectally, intranasally or as aninhalant, for example.

When taken orally, the oleocanthal formulation may be in the form of alollipop, quick-dissolving film, tablet, syrup, liquid, liqui-gel,capsule, or the like.

The amount of oleocanthals in the preparation may be adjusted by aphysician of skill in the art for suitable dosages for adults orpediatric use, or by a veterinarian of skill in the art for use invarious animals. The dosage of drug may be determined based on theweight of the subject or based on surface area. Any method ofdetermining proper dosages is acceptable.

The oleocanthals are preferably formulated with a pharmaceuticallyacceptable diluent, excipient or carrier (collectively referred toherein as “carrier” materials) as described above.

(2) Counter-Irritant for Sore Throat:

The oleocanthals of the invention are useful as counter-irritants forsore throat which may accompany a cold or flu, for example. Theoleocanthal may be applied in combination with other ingredients forsore throat relief or may be provided as the sole active ingredient. Theoleocanthal-based sore throat formulations may be in the form of atablet, lozenge, lollipop, chewing gum, or throat spray. The formulationmay be prepared and packaged by any means known in the art.

For example, solid dosage forms may contain other ingredients known insuch dosage forms such as acidity regulators, opacifiers, stabilizingagents, buffering agents, flavorings, sweeteners, coloring agents, andpreservatives. For example, a lozenge may be prepared as by heating thelozenge base (e.g., a mixture of sugar and liquid glucose) under avacuum to remove excess water and the remaining components are thenblended into the mixture. The resulting mixture is then drawn intodesired shape. The lozenges are cooled, and packaged into suitablepackaging. Lozenges will normally be sucked by the patient to releasethe oleocanthal. Chewable solid dose formulations may be made by themethods used to prepare chewable candy products or chewing gums. Forexample, a chewable solid dosage form may be prepared from an extrudedmixture of sugar and glucose syrup to which the oleocanthal has beenadded with optional addition of whipping agents, humectants, lubricants,flavors and colorings. (See Pharmaceutical Dosage Forms: Tablets, Volume1, Second Edition edited by H A Lieberman, L Lachman and J B Schwartzpublished in 1989).

Spray formulations may be prepared by dissolving or suspending theoleocanthal in a liquid medium which may also contain other ingredientssuch as stabilizing agents, buffering agents, flavorings, sweeteners,coloring agents, and preservatives. For example, a spray may be preparedby dissolving water soluble components in water and non-water solubleingredients in a co-solvent (e.g., alcohol). The two phases are thenmixed and the resulting mixture filtered and placed into dispensingcontainers. The dispensing containers may be fitted with a metered,manually-operated spray mechanism or the dispenser may contain apressurized propellant and be fitted with a suitable dispensing valve.

(3) Nasal Decongestant:

The oleocanthals of the invention are useful as a nasal decongestant.The oleocanthal may be applied in combination with other nasaldecongestants or may be provided as the sole active ingredient. Theoleocanthal-based nasal formulations may be in the form of a lavage ornasal mist. The formulation may be prepared and packaged by any meansknown in the art for nasal lavages and mists.

(4) Antioxidant:

Oleocanthals are believed to have anti-oxidant activity and as such maybe used to treat or prevent various conditions including cancer. Theoleocanthals may also be used to promote wound healing, either byapplication directly onto wounds, or as a coating or impregnation ofbandages, sutures and the like.

The antioxidant effects of oleocanthals may also be exploited in theformulation of cosmetics. The compositions can protective of skin orhair or as an anti-solar composition. In accordance with the inventionthe compound of formula (I), and preferably oleocanthal is generallypresent in an amount ranging from 1 to 1,000 mg. In some embodiments,oleocanthal or an oleocanthal derivative is present in an amount ofabout 5 to 800 mg. In other embodiments, oleocanthal or an oleocanthalderivative is present in an amount of about 10 to 750 mg. In otherembodiments, oleocanthal or an oleocanthal derivative is present in anamount of about 25 to 600 mg. In other embodiments, oleocanthal or anoleocanthal derivative is present in an amount of about 50 to 500 mg. Incertain embodiments, the oleocanthal or derivative thereof is present in1, 5, 10, 20, 25, 50, 75, 100, 125, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 or 1,000 mg.

In the compositions according to the present invention, the compound offormula (I) acts as an antioxidant agent. These compositions can becapillary compositions such as hair lacquers, hair setting lotions orhair treating or disentangling lotions, shampoos, coloring shampoos,hair dye compositions, makeup products such as nail enamels, skintreating creams and oils, foundations, lipsticks, compositions for thecare of the skin such as bath oils or creams as well as any othercosmetic composition capable of exhibiting, because of their components,oxidation stability problems during storage.

(5) Pain Relief:

The oleocanthals of the invention may be used as to treat and preventpain. The compounds are useful for the relief of pain associated with avariety of conditions including, but not limited to influenza or otherviral infections, common cold, low back and neck pain, dysmenorrhea,headache, toothache, sprains and strains, myositis, neuralgia,synovitis, arthritis, including rheumatoid arthritis, degenerative jointdiseases (osteoarthritis), gout and ankylosing spondylitis, bursitis,burns, injuries, cancer and for pain associated with surgical and dentalprocedures.

(6) Anti-Inflammatory:

The oleocanthals of the invention may be used as anti-inflammatoryagents. The oleocanthals may be used in a method for treating orpreventing diseases marked by inflammation, including but not limited topsoriasis, cancer, asthma, allergic rhinitis, respiratory distresssyndrome, inflammatory bowel disease, Crohn's disease, gastritis,irritable bowel syndrome, ulcerative colitis, migraine, periarteritisnodosa, thyroiditis, aplastic anemia, Hodgkin's disease, scleroderma,type I diabetes, myasthenia gravis, multiple sclerosis, sorcoidosis,ischemic kidney disease, nephrotic syndrome, Bechet's syndrome,polymyositis, gingivitis, conjunctivitis, vascular disease myocardialischemia, heart disease, stroke, and hypertension.

(7) Micro-G Protein and Associated Kinase Inhibitor:

The oleocanthals of the invention may also be formulated for treatmentor prevention of the development of Aβ42 associated Alzheimer's plaquesand tangles in a manner similar to that found for non-steroidalanti-inflammatory drugs such as ibuprofen. Without wishing to be boundby any particular theory of operation, it is believed that ibuprofen,and oleocanthal inhibit micro-G proteins and associated kinases, forexample Ras and Rock, which have been associated with the development ofAβ42 associated plaques and tangles in the brains of Alzheimer'spatients. Oleocanthals also acts to inhibit γ-secretases and alterpresenilin conformations of which both activities are associated withreducing Aβ42 associated Alzheimer's plaques and tangles.

It is believed that certain non-steroidal anti-inflammatory drugsinhibit γ-secretases without significantly altering other activities inthe Aβ amyloid precursor protein (APP) processing pathway. In patientswith certain mutations in APP and all mutations known for presenilin,APP is processed such that there is a large increase in the amount of aproteolytic fragment of 40-42 residues (Aβ42) (Weggen et al. (2001)Nature 414 (8):212). Certain NSAIDs appear to have an effect of reducingthe production of Aβ42 by a mechanism that is independent of thecyclooxygenase activity associated with the anti-inflammatory activityof the NSAIDs. It has been shown that for many NSAIDs, which areadministered as racemic mixtures of the active compounds, that aspecific enantiomer (the S-enantiomer) appears to be responsible for theinhibition of cyclooxygenase activity, and hence the anti-inflammatoryeffect (Weggen et al. (2001) Nature 414 (8):212). It has also been shownthat the R-enantiomer of the NSAIDs may mediate reduction of Aβ42production and may be responsible for the decreased risk in Alzheimer'sand cognitive impairment seen with long term use of NSAIDs (Morihara etal. (2002) J. Neurochem. 83:1009-1012).

Also correlating with lower risk of developing Alzheimer's and cognitiveimpairment is the so-called Mediterranean diet, which is typically highin consumption of, among other things, olive oil. Thus, the observationmade herein of the association with the organoleptic properties ofoleocanthal and the similarity to ibuprofen and the observationsassociated with long term use of NSAIDs and dietary intake of olive oilsuggest that oleocanthal may be used for the treatment and prevention ofneurodegenerative disorders (e.g., Alzheimer's and other cognitiveimpairment associated with amyloid plaques and tangles). The treatmentand prevention of such neurodegenerative disorders may be performedusing a racemic mixture of oleocanthal, or may be using one of thepurified enantiomers of oleocanthal.

(8) For Oral Surgery and Oral Irradiation Treatment of Cancer:

The oleocanthals of the present invention are also useful as treatmentsfor use in conjunction with oral surgery and oral irradiation treatmentof cancer. While not wishing to be bound by any particular theory ofoperation, it is believed that the oleocanthals, with their attendantanti-inflammatory activity, act to inhibit the inflammation that occursin the oral cavity as a result of surgery or oral irradiation. Theoleocanthals may be formed as an oral rinse which can be administeredbefore the procedure, after the procedure, or during the procedure, or acombination of these treatment regimens. The amount of oleocanthal inthe rinse is a therapeutically effective amount which is readilydetermined by one of skill in the art.

D. Animal Repellant:

It is believed that oleocanthals, with their organoleptic qualities, areuseful as animal repellents. The compounds may be used to repelcarnivorous and omnivorous animals and birds, including domestic cats,rodents, raccoons, dogs, other canids such as coyotes.

The method of this invention comprises applying an effective, repellentamount of the oleocanthals, either alone or in combination with asuitable carrier, to the locus from which the animals are to berepelled. Suitable carriers would include liquid diluents such as water,hydrocarbons, alcohols, emulsifiers and other liquids generally found inhousehold spray formulations or pharmaceutical preparations so as to beacceptable from a human safety viewpoint. Inert solid carriers such asstarches may also be of use, and it might be desirable to incorporatethe compounds into a controlled-release formulation.

It may be desirable to apply the oleocanthals to containers fordiscarded edible refuse, such as metal or plastic garbage cans, plasticbags, paper and cardboard boxes and the like. Further, the repellentcompounds disclosed herein might be incorporated into variouspotentially-edible compositions which, if consumed, could injure or killan animal. An example of such a composition would be liquid antifreeze.

Another aspect of this invention provides methods for repelling birdsfrom consuming or utilizing a material otherwise susceptible toconsumption or utilization by birds, comprising providing to thematerial an avian repellent amount of at least one oleocanthal.

Liquid carriers may be employed and the repellent may be sprayed on thematerial. See e.g., U.S. Pat. No. 2,967,128 which patent is incorporatedby reference as if fully set forth herein. The compound may be dispersedin the liquid from which the birds are to be repelled. The repellent maybe at least partially trapped in a solid vehicle to improve itspersistency such as disclosed in U.S. Pat. No. 4,790,990. The vehiclemay be a modified starch, oil or polymer which at least partiallyencapsulates, emulsifies or substantially uniformly disperses theaversive agent. The repellent compound and vehicle may be dispersedthroughout solids consumed by avian species to reduce the likelihoodthat they will eat the treated edible.

Certain embodiments of the present invention are directed to methods ofrepelling birds from consuming or utilizing non-potable liquids such asindustrial or agricultural waste water, mine tailing ponds, andfreestanding water on artificial surfaces like airport runways andparking lots. “Non-potable” refers to liquids or aquatic habitatswherein said liquid may be consumed or utilized by birds to thedetriment of man or the birds.

E. Discovery

Knowledge of the absolute structure of oleocanthal allows theidentification of the oleocanthal receptor and related genes. Screeningassays for receptors, well-known in the art, may be employed todetermine the oleocanthal receptor. Tissue from the back of the throat,known to interact with oleocanthal may be isolated and subjected tovarious assays to determine the binding of oleocanthal to cells and themolecular signaling pathway of oleocanthals.

Labeled oleocanthal may be used in tissue binding studies to determinethe cell types that contain a presumptive oleocanthal receptor. Cellsthat have bound labeled oleocanthal may be visualized by any methodknown in the art. For example, but not by way of limitation, oleocanthalmay be labeled with a radiolabel (e.g., ¹²⁵I, ³⁵S, ³²P, ³³P, ³H), afluorescence label, a chemiluminescent label, an enzymatic label, or animmunogenic label. In other embodiments, luminescent or fluorescentmolecules may be conjugated to the oleocanthal molecule. The labeledoleocanthal may be allowed to bind to cells in situ and visualized undera microscope. Alternatively, cells in suspension may be labeled with thelabeled oleocanthal and labeled cells may be separated from unlabeledcells by flow cytometry or using a sorter, such as afluorescence-activated cell sorter (FACS). Labeled cells may becollected for subsequent genetic analysis, for example.

In some embodiments, a molecule is conjugated to oleocanthal that allowsthe conjugated oleocanthal to be cross-linked to its receptor uponbinding. This may be performed by any means known in the art. Thereafterthe cross-linked receptor may be isolated from the cells, purified andsubjected to N-terminal amino acid sequencing. With the identity of theN-terminal amino acids, degenerate oligonucleotides may be synthesizedbased on the possible combinations of oligonucleotides encoding theamino acid sequence and the oligonucleotides may be used in various waysto identify the gene encoding the oleocanthal receptor. In someembodiments, the degenerate oligonucleotides are used to probe genelibraries. The gene library may a library formed from animal cells,particularly human cells, or it may be a specific cell-type library fromanimal cells known to be responsive to oleocanthal. In otherembodiments, the library may be a subtractive library formed by removingcommonly expressed genes from oleocanthal-responsive andoleocanthal-unresponsive cells, such that the library consists of asubset of genes reflecting unique sequences of theoleocanthal-responsive cells. In another embodiment, the degenerateoligonucleotides are paired with a second set of oligonucleotides toallow rt-PCR amplification of polynucleotides containing the sequencesencoding the amino acid sequence of the oleocanthal receptor. Suchsecond set of oligonucleotides may include, for example, oligo-dT whichanneals to poly-adenosine tracts of mRNA. The rt-PCT reaction may beperformed on RNA extracted from oleocanthal responsive cells. Themethods and techniques for such genetic analysis are well-known in theart and may be found in the references and texts referred to herein.

Further aspects of the invention are exemplified below, however, theexamples are merely illustrative of the invention and the scope of theinvention is not to be limited thereto or thereby.

EXAMPLES Example 1 Isolation of Deacetoxydialdehydic LigstrosideAglycone “Oleocanthal”

A. Synthesis of Oleocanthal

Retrosynthetically, we envisioned both enantiomers of (1) to derive fromthe enantiomeric forms of cyclopentanediols (7) via oxidative cleavageof the diol moiety (Scheme 1). The requisite cyclopentanediols (7) inturn would be prepared from cyclopentanones (+)- and (−)-(10), viaalkylation to introduce stereoselectively the side chain from the convexface, followed by stereoselective Wittig ethylnation and removal of theacetonide moiety (Scheme 1).

(5) Initially (+)- and (−)-cyclopentanones (10) were prepared via thesulfoximine and/or enzymatic protocols introduced and developed byJohnson (Johnson, C. R. and T. Penning (1988) J. Am. Chem. Soc.110:4726-4735; Johnson, C. R. (1998) Acc. Chem. Res. 31:333-341).Although effective on modest scale (10-100 mg), the requirement for gramquantities of the oleocanthals demanded that we secure for more scalableroutes to (10). Towards this end, we optimized a hybrid of syntheticapproaches (Moon, H. et al. (2002) Tetrahedron: Asym. 13 (11):1189-1193;Jin, Y. et al. (2003) J. Org. Chem. 68 (23):9012-9018; Yang, M. (2004)J. Org. Chem. 69 (11):3993-3996; Palmer, A. et al. (2001) Eur. J. Org.Chem. 66 (7):1293-1308; Paquette, L. and S. Bailey (1995) J. Org. Chem.60:7849-7856) as outlined in Scheme 2. Importantly, both enantiomers of(10) could be prepared in multi-gram quantities in 7 steps, with anoverall efficiency of 40% from inexpensive D-(−)-ribose. Key elements ofboth sequences entailed vinyl Grignard addition to the enantiomers ofaldehyde (12), followed in turn by ring closing metathesis (RCM), PCCoxidation and hydrogenation (Scheme 2).

Alkylation of (+)- and (−)-cyclopentanone (10) with methyl bromoacetatewas then anticipated to proceed from the less hindered convex face ofthe bicyclic skeleton to install the side chain in a stereoselectivefashion. Initial attempts however to alkylate (−)-(8) with methylbromoacetate employing LDA in the presence of HMPA furnished only acomplex mixture containing only trace amounts of (−)-(16). Neitheraddition of Cu(I) (Johnson, C. R. and T. Penning (1988) J. Am. Chem.Soc. 110:4726-4735) reportedly to suppress side reactions, nor the useof the corresponding tin enolate [generated by treatment of (−)-(10) inTHF with LDA, followed by HMPA and tributyltin chloride (Suzuki, M. etal. (1985) J. Am. Chem. Soc. 107:3348; Nishiyama, H. et al. (1984)Tetrahedron Lett. 25:223)] improved the situation. Alkylation of thezinc enolate of (−)-(10) [generated by treatment of (−)-(10) in THF with1.1 eq. LHMDS, followed in turn by HMPA (3.0 eq.) and dimethyl zinc(Morita, Y. et al. (1989) J. Org. Chem. 54:1787-1788) (1.0 eq.)] withmethyl bromoacetate, however consistently furnished (−)-(16) in 55-60%yield as a single diastereomer (this reaction was fairly clean exceptsome baseline materials. Using t-butyl bromoacetate instead of methylbromoacetate did not improve the yield) (Scheme 3).

Wittig ethylnation of (−)-(16) was next achieved withethyltriphenylphosphine bromide. Best results were obtained employingLDA as the base at −45° C. Although excellent stereoselectivity (ca.,10:1 E:Z) favoring the E-isomer (−)-(17) was achieved, the yield wasonly modest (42%), presumably due to the ease of enolization of (−)-(16)(Edmunds, M. “The Wittig Reaction” In MODERN CARBONYL OLEFINATION,Takeda, Ed., John Wiley & Sons, New Jersey, 2004). Interestingly, thestereoselectivity varied dramatically with reaction temperature. At 0°C., the E:Z selectivity was 3.3:1, while at room temperature theselectivity was 1.6:1. Assignment of the E geometry of the olefin wasbased on NMR NOE analysis (Scheme 4).

Hydrolysis of ester (−)-(17) (LiOH/THF/H₂O) next afforded acid (−)-(18),which was subjected to Mitsunobu esterification (Mitsunobu, O. (1981)Synthesis 1-28) with 4-hydroxyphenethyl alcohol to furnish phenol(−)-(19) in 92% yield. As expected, the Mitsunobu reaction proceededwith complete chemoselectivety at the primary hydroxyl (Appendino, G. etal. (2002) Org. Lett. 4:3839-3841). Completion of the synthesis of(−)-oleocanthal (1) was then achieved via liberation of the vicinal diolmoiety (4N HCl/acetonitrile), followed by oxidative cleavage (NaIO₄);(−)-oleocanthal (1) was identical in all respects (e.g., ¹H and ¹³C NMR,IR and HRMS) with an authentic sample isolated from virgin olive oil,the latter possessing spectral data identical to that reported in theliterature (Montedoro, G. et al. (1993) J. Agric. Food Chem.41:2228-2234). The structural assignment of (1) was also confirmed byCOSY NMR analysis. Synthetic (−)-(1) displayed a small negative opticalrotation ([α]²⁵ _(D) −0.78, c=0.9, CHCl₃) identical to that obtainedfrom a sample isolated from virgin olive oil ([α]²⁵ _(D) −0.9, c=2.0,CHCl₃). Thus the stereochemistry of (−)-oleocanthal (1) is 3S, 4E. Theenantiomer of the natural product (+)-(1) was prepared via a similarreaction sequence beginning with (+)-(10) to furnish (+)-1 ([a]²⁵ _(D)+0.73, c=0.55, CHCl₃) (Scheme 5).

In summary, an effective, scalable synthesis of both enantiomers ofoleocanthal (1) has been achieved, each hi 13 steps (7% overall yield)from inexpensive (D)-(−)-ribose, requiring only 6 chromatographicseparations. The structural similarity of oleocanthal to a number ofrelated natural products (Somanadhan, B. et al. (1998) Planta Medica64:246-50; Takenaka, Y. et al. (2002) Chem. & Pharm. Bull. 50(3):384-389; Takenaka, Y. et al. (2002) Phytochemistry 59 (7):779-787)suggests that the synthetic approach presented here should also beapplicable to their construction.

B. Functional Studies of Oleocanthal

This restricted throat irritation of oleocanthal is remarkably similarto that elicited by ibuprofen. Due to the observed organolepticsimilarity, we isolated and then synthesized oleocanthal from olive oil.Sensory and chemical evaluation of 10 commercially available olive oilsrevealed a strong positive relationship between throat irritationintensity and oleocanthal concentration. Cyclooxygenase and lipoxygenaseassays with synthesized oleocanthal demonstrated that it is a NSAID withan anti-inflammatory profile strikingly similar to that of ibuprofen, inaccordance with its sensory properties. Oleocanthal may play asignificant role in the well-known health benefits associated with adiet high in olive oil. Moreover, identification of otherpharmacologically important compounds is hereby facilitated by attentionto similarities of sensory properties.

Recent studies in our laboratories have demonstrated that ibuprofen, aswell as some other non-steroidal anti-inflammatory drugs (NSAIDs), havethe unusual sensory property of stinging almost exclusively in thethroat, unlike for example, capsaicin and piperine that also burn themouth and lips. While tasting newly-pressed Sicilian olive oil, it wasobserved that the throat-irritating sensation appeared identical to thatof ibuprofen. Indeed, high quality extra virgin olive oils are oftencharacterized by a stinging or burning sensation akin to that felt whenswallowing ibuprofen. With olive oil, this sensation often elicits asmall cough or throat-clearing when olive oil is swallowed neat. Oliveoil enthusiast categorize oils as 0, 1, 2 or 3 cough oils with thehigher numbers being superior. The entity responsible for this sensoryproperty has recently been reported to be deacetoxy-dialdehydicligstroside aglycone, one of many polyphenols found in olive oil.

Based on their similar oro-sensory properties, we reasoned thatoleocanthal might also share pharmacologic properties of ibuprofen. Totest this thesis, we first had to verify and definitely prove theidentity of oleocanthal. This required development of an efficientanalytical method for isolating and quantifying it. Two approaches toverify the identity of the compound and its properties were taken.First, we undertook psychophysical experiments with oleocanthal,correlating the amount of identified compound with the degree of burn incommercial olive oils. Second, we synthesized oleocanthal and tested thepsychophysical properties of the synthesized material. Finally, toexamine oleocanthal for pharmacological activity that might mimicibuprofen, cyclooxygenase, lipoxygenase and lipid peroxidation assayswith synthetic material were conducted.

To isolate and purify oleocanthal, we employed a systematicsensory-directed approach. That is, we used taste analysis as a tool tomonitor the presence of the throat-irritation compound in each step ofan isolation and purification procedure similar to that used by Andreweset al. Briefly, the irritant was first extracted from olive oil withmethanol/water (80/20, v/v). The phenolic extract was separated into 15fractions, only one of which was irritating, using a reversed-phase HPLCmethod. To obtain pure material, we pre-fractioned the olive oilphenolic extract on a C18 solid phase extraction cartridge. Retentioninformation about the throat-irritating principal from the HPLC methodallowed us to separate it from the majority of the other co-extractedphenolic compounds using methanol and water solvent mixtures at threedifferent ratios of eluting solvents. HPLC analysis of the throatirritating fraction revealed the presence of several unresolvedcompounds. A new HPLC gradient was thus developed and only onewell-resolved peak was throat-irritating. A detailed NMR (1D and 2D)analysis was conducted with this material. Although ¹H-NMR spectraindicated the presence of minor impurities, the structure of the majorcompounds was readily identified to be 2-(4-hydroxyphenyl)ethyl,4-formyl-3-(2-oxoethyl)-4-hexenoic acid ester, the deacetoxy-dialdehydicligstroside aglycone, as first identified in olive oil by Montedoro andrecently reported as the throat irritant. Optical rotation measurementsof oleocanthal revealed the natural enantiomer to be levorotary.

Olive oils differ markedly in their ability to elicit throat irritation.If oleocanthal is primarily responsible for this sensory property thereshould be a positive relation between compound concentration and degreeof throat irritation. To test this hypothesis, we purchased 10 differentolive oils with widely varying degrees of throat irritation based oninformal evaluation. The amount of oleocanthal in each was thenquantified. The compound was extracted from small amounts of each of the10 oils (1 g) by hexane-acetonitrile (liquid-liquid) extraction. Thesolvent extract was analyzed by reverse-phase HPLC with UV detection at278 nm. Oleocanthal was chromatographically separated from the otherextracted compounds with an elution gradient of acetonitrile and water.All analyses were done in duplicate using solutions of pure, isolatedoleocanthal as the external standard. When the compound was latersynthesized, this was also used as a standard to confirm these methods.Overall, the reproducibility was high (RSD=4.7%), recovery was good(95%), the calibration curve was linear (r²=0.9999) and the limit ofquantitation was <1 ppm.

The degree of throat irritation of these 10 oils was quantified by 17volunteers. Each subject was tested only 2 times per day with twodifferent olive oils samples with 1-2 hours separating each test sincethe irritation may be sensitive to shorter inter-trial intervals.Subjects wore nose clips to eliminate olfactory cues. Tasting consistedof placing approximately 3.5 ml of olive oil in the mouth, holding itthere for 3 seconds and then swallowing it in two aliquots so as toinsure the throat would be stimulated. After 45 seconds passed, subjectswere asked to rate the peak throat irritation sensitivity using thegeneral labeled magnitude scale, a sensory scale developed to generatemagnitude estimation-like quality data. Each subject was tested twicewith all ten oils.

The concentrations of oleocanthal in the 10 olive oils and their degreeof throat irritation proved statistically significant (r=0.90; FIG. 2)providing additional evidence that oleocanthal is responsible for themajority of the throat irritation in the olive oils tested.

These studies strongly implicate oleocanthal as the major throatirritating compound in olive oil. Nevertheless, as noted by Andrewes etal., co-elution of a minor component or mixture of components causingthe burning sensation cannot be eliminated as a possible source ofirritation without completing a de novo total synthesis followed byorganoleptic analysis. Since the structure of oleocanthal possesses astereospecific center, we synthesized both enantiomers from readilyavailable D-ribose. The synthesis of both (+) and (−)-oleocanthalrequired 13 steps as outlined for the recovery of levorotary(−)-enantiomer in FIGS. 3 and 4. Both syntheses proved scalable,proceeding in 7% overall yield and thereby providing ample material forsensory and pharmacological evaluation. The levoratory (−)-enantiomer ofsynthetic oleocanthal displayed the same sign and magnitude of theoptical rotation as the natural material. Thus the absolutestereodirection of variant (1) is as depicted in FIG. 2.

Three individuals experienced in tasting olive oils and ibuprofen, usinga standard 2 alternative forced-choice method, evaluated the syntheticcompound (the natural (−)-isomer only) dispersed in non-irritating cornoil at approximately twice the concentration found in Falconaro oliveoil, the most potent olive oil we have evaluated (FIG. 1).

Testing was double-blind and each was exposed to three sets of twosamples, one of which was added synthesized oleocanthal and the otherserved as the blank control. The task was to indicated which of the pairwas more irritating. Each of the three evaluators correctly identifiedthe sample on each of the three presentations (9 of 9 correct, p<0.01)and all three identified the distinct back of the throat irritation withthe cough-eliciting sensation characteristic of both olive oil andibuprofen. As predicted, the throat irritation of synthetic(−)-oleocanthal was identical to oleocanthal isolated from premium oliveoil. Importantly, the effect was dose dependent (FIG. 2 open triangles,dashed line). Ten subjects were tested with non-irritating commercialcorn oils presented neat and mixed with either synthesized(−)-oleocanthal or the bitter agent sucrose octaacetate (SOA)(Sigma-Aldrich). The addition of SOA enabled forced-choice trials to beconducted without revealing to subjects the identity of the irritatingsamples due to bitterness or other non-irritating cues. (−)-Oleocanthalwas tested at the highest concentration identified in the ten ratedoils, 200 μg/ml, and at one half and whole log steps more dilute 63.25and 20 μg/ml. SOA was added to the corn oil (4×10⁻⁴, 1×10⁻⁴, 5×10⁻⁵ M)to intensity match the irritation of the three levels of(−)-oleocanthal. Subjects participated in two-alternative forced-choice(2AFC) trials (four trials at every concentration for each subject) andin intensity ratings sessions (four ratings per each oil). For the 2AFCtrials subjects were presented with two 3.0 ml corn oil samples withmatching intensities of SOA and (−)-oleocanthal in ascending order, andwere required to sample oils as described above. While blind to stimulusposition, subjects were asked two questions on each trial, “Which of thetwo oils was more irritating in the throat?” and “Which one was morebitter?” At the 20 μg/ml & 5×10⁻⁵ M level most subjects reported on sometrials that the same oil was both the more irritating and the morebitter of the two. This demonstrates that participants were willing toselect the one oil as stronger on both traits within a trial. Subjectsperformed at chance when selecting among two unadulterated corn oils,when the correct choice was randomly assigned prior to testing. At 20μg/ml subjects were correct 24 out of 40 trials, indicating that thisconcentration is near detection threshold levels in corn oil. The othertwo concentrations were correct 39/40 and 40/40 trials. For theintensity rating trials subjects were presented with all eight oils inascending order, counterbalanced for stimulus order and asked to ratethe throat irritation and bitterness of every oil on a general labeledmagnitude scale.

Assuming the quality and locus of irritation provides a signal ofpharmacological activity, then oleocanthal should mimic at least some ofthe pharmacological properties of ibuprofen, a potent modulator ofinflammation. To test this we chose to evaluate inhibition ofcyclooxygenase (COX) and lipoxygenase (LO), two enzymes central to theinflammatory process. Ibuprofen is a potent COX-1 and COX-2 inhibitorbut does not inhibit lipoxygenase. The concentration dependence ofoleocanthal for inhibition of ovine COX-1, human recombinant COX-2 andsoybean 15-lipoxygenase activities was measured using commerciallyavailable kits (Cayman Chemicals). Indomethacin was used as a positive(inhibitory) control in the cyclooxygenase assays andnordihydroguaracetic acid (NDGA) and caffeic acid were used as positive(inhibitory) controls in the lipoxygenase assays. Both enantiomers ofoleocanthal, exhibited a dose-dependent inhibition of both COX-1 andCOX-2 activities, with no effect on lipoxygenase activity, much asobserved with ibuprofen (Table 1). The calculated IC₅₀ (least squaresregression analysis of inhibition vs. concentration) for oleocanthal (−)was 21.4 μm and 29.4 μm for COX-1 and COX-2, respectively. The IC₅₀ foroleocanthal (+) was 27.9 μm and 40.5 μm for COX-1 and COX-2,respectively. In these experiments, both enantiomers of oleocanthal weremore potent at equimolar concentrations than ibuprofen in inhibitingCOX-1 and COX-2. Both enantiomers of oleocanthal inhibited theperoxidation of serum lipids induced by metal ions in vitro to a similardegree as equimolar alpha-tocopherol (data not shown). Thus, oleocanthalexhibits antioxidant activity comparable to alpha-tocopherol and has anarachidonic acid inhibitory profile (cyclooxygenase inhibition withoutlipoxygenase inhibition) indicating that both enantiomers of oleocanthalare classic NSAIDs, with potency superior to that of ibuprofen.

Taken together, these data are consistent with our hypothesis that thethroat irritating compound in olive oil is an ibuprofen-likeanti-inflammatory agent. Importantly, the oleocanthal results provide anexample of how sensations from the mouth may serve as an in vivopharmacological assay. These results further suggest an additional basisfor the health benefits of olive oil consumption have been attributed toa combination of the lipid profile, the antioxidant activity of many ofthe polyphenols present and the anti-inflammatory agents that inhibitlipoxygenase. We suggest here an additional benefit: long-termconsumption of oleocanthal, with anti-inflammatory ibuprofen-likeactivity may enhance health and well-being. Assuming that an olive oilconsumer in the high normal range ingest about 50 g of olive oil per dayand that this oil contains up to 200 μg/ml of oleocanthal, the personwould then consume approximately 10 mg/day. Although this dose isrelatively low (−10% of the dosage of ibuprofen recommended for adultpain relief), chronic low doses of other COX inhibitors (e.g., aspirin)are known to have important health benefits, chiefly a reduction inheart attack risk and at slightly higher doses a reduction in both heartattack and stroke risk.

In addition to anti-inflammatory activity, ibuprofen has recently beenshown to have a COX-independent ability to decrease the highlyamyloidogenic AB42 peptide, perhaps accounting for epidemiologicevidence that Alzheimer's disease. Thus, it would be important todetermine whether oleocanthal has similar activity.

The initial hypothesis that the throat irritating compound in olive oilmight have pharmacological activity was based on the oro-sensorysimilarities of ibuprofen and olive oil. This implies a similar sensorymechanism but exactly how ibuprofen (or oleocanthal) elicits almostexclusive throat irritation remains elusive. One possible explanation isthat there is a currently unknown receptor system that is responsible toboth ibuprofen and olive oil. Alternatively, or additionally, bothcompounds could have particularly easy access to free nerve endings inthe throat, but why this would occur preferentially in the throat isunknown. It is also unclear why other lipophilic irritants such aslactic acid or capsaicin would not stimulate the throat exclusively aswell, if the mechanism were simply one of ready access to free nerveendings. Elucidation of the sensory mechanism may assist in determiningthe common pathway for the anti-inflammatory activities of thesemolecules, or vice versa. The sensory properties of foods, spices andflavors may provide clues to pharmacological activity and thus serve notonly to provide pleasure but also to enhance health.

TABLE 1 Percent inhibition of COX-1, COX-2 and 15-LO by Oleocanthal (−),(+) Concen- tration Agent (uM) COX-1 COX-2 15-LO Oleocanthal (−) 10083.5 ± 3.5 70.9 ± 8.6 0.4 ± 0.8 25 56.1 ± 3.2 56.6 ± 9.5 0.0 ± 0.0 724.6 ± 7.3 14.5 ± 2.3 0.0 ± 0.0 Oleocanthal (+) 100  68.0 ± 15.2 69.6 ±3.9 3.5 ± 5.5 25 54.5 ± 4.6  41.3 ± 15.9 0.7 ± 1.0 7 24.6 ± 7.5  6.1 ±4.2 0.0 ± 0.0 Ibuprofen 25 17.8 ± 2.3 12.7 ± 3.6 0.2 ± 0.3 7 0.0 1.3n.d. Indomethacin 25 45.8 ± 4.4  77.6 ± 10.2 0.1 ± 0.9 7 33.0 ± 6.1 71.6± 7.3 0.5 ± 0.1 NDGA 25 n.d. n.d. 63.1 ± 0.8  7 n.d. n.d. 52.5 ± 1.1 Caffeic Acid 25 n.d. n.d. 25.2 ± 2.2  7 n.d. n.d. 19.8 ± 1.3  * Data arepresented as mean % inhibition ± SEM for three independent experiments.N.d. = not determined.

Example 2 Structure Activity Relationship (SAR) Study

A Structure Activity Relationship (SAR) Study may be conducted todetermine the functional relative activities of oleocanthal derivatives.As shown in FIG. 5, a compound having the structure:

is reacted with a compound selected from the following:

to produce oleocanthal derivatives. These compounds are then tested foractivity as described above. Relative efficacies and potencies of theoleocanthal derivatives may be assigned to each compound andstructural-functional information may be derived for rational drugdesign of oleocanthals.

1. A method of synthesizing a purified (−) enantiomer of a compoundhaving the formula:

comprising (a) converting D-ribose into a compound of Formula I with astrong acid in a suitable solvent; (b) contacting the compound ofFormula I with a halogenation reagent followed by metal-halogen exchangeinduced ring opening to yield a compound of Formula IIa; (c) contactingthe compound of Formula IIa with CH₂═CH—MgBr in a suitable solvent toyield a compound of Formula IIIa; (d) contacting the compound of FormulaIIIa with Grubbs catalyst in a suitable solvent followed by oxidation toyield a compound of Formula IVa; (e) contacting the compound of FormulaIVa with hydrogen, palladium catalyst in a suitable solvent to yield(−)-cyclopentanone (Formula Va); (f) contacting the (−)-cyclopentanonewith lithium hexamethyldisilazide in a suitable solvent followed byhexamethylphosphoramide (HMPA) dimethyl zinc and alkyl bromoacetate toyield a compound of Formula VIa; (g) subjecting the compound of FormulaVIa to a Wittig ethylnation using ethyltriphenylphosphine bromide atreduced temperature; (h) hydrolyzing the ester to yield a compound ofFormula VIIIa; (i) contacting the compound of Formula VIIIa with4-hydroxyphenethyl alcohol under conditions to perform an esterificationto yield a compound of Formula IXa; (j) liberating the vicinal diolmoiety; and (k) performing an oxidative cleavage to yield(−)-oleocanthal (Formula Xa).
 2. A method of synthesizing a purified (+)enantiomer of a compound having the formula:

comprising: (a) converting D-ribose into a compound of Formula XI with astrong acid in acetone; (b) contacting the compound of Formula XI with amethyltriphenylphosphine bromide, followed by oxidative cleavage toyield a compound of Formula IIb; (c) contacting the compound of FormulaIIb with CH₂═CH—MgBr in a suitable solvent to yield a compound ofFormula IIIb; (d) contacting the compound of Formula IIIb with Grubbscatalyst, in a suitable solvent, followed by oxidation to yield acompound of Formula IVb; (e) contacting the compound of Formula IVb withhydrogen, palladium catalyst in a suitable solvent to yield(+)-cyclopentanone (Formula Vb); (f) contacting the (+)-cyclopentanonewith lithium hexamethyldisilazide in a suitable solvent followed byhexamethylphosphoramide (HMPA), dimethyl zinc and methyl bromoacetate toyield a compound of Formula VIb; (g) subjecting the compound of FormulaVIb to a Wittig ethylnation using ethyltriphenylphosphine bromide atreduced temperature; (h) hydrolyzing the ester to yield a compound ofFormula VIIIb; (i) contacting the compound of Formula VIIIb with4-hydroxyphenethyl alcohol under conditions to perform an esterificationto yield a compound of Formula IXb; (j) liberating the vicinal diolmoiety; and (k) performing an oxidative cleavage to yield(+)-oleocanthal (Formula Xb).
 3. A compound comprising the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅; wherein said compound is other than oleocanthal.
 4. A methodof inhibiting COX-1, COX-2, COX-3 or lipoxygenase comprisingadministering an effective amount of a compound having the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅.
 5. The method of claim 4 wherein said compound is oleocanthal.6. The method of claim 5 wherein said oleocanthal is the (−)-enantiomer.7. The method of claim 5 wherein said oleocanthal is the (+)-enantiomer.8. An anti-inflammatory composition comprising a therapeuticallyeffective amount of a compound having the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅; and a pharmaceutically acceptable carrier.
 9. The compositionof claim 8 wherein said compound is oleocanthal.
 10. The composition ofclaim 9 wherein said oleocanthal is the (−)-enantiomer.
 11. The methodof claim 9 wherein said oleocanthal is the (+)-enantiomer.
 12. Anantioxidant composition comprising a therapeutically effective amount ofa compound having the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅; and a pharmaceutically acceptable carrier.
 13. The compositionof claim 12 wherein said compound is oleocanthal.
 14. The composition ofclaim 13 wherein said oleocanthal is the (−)-enantiomer.
 15. The methodof claim 13 wherein said oleocanthal is the (+)-enantiomer.
 16. A methodof enhancing the flavor of food comprising adding an effective amount ofa compound of the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅.
 17. The method of claim 16 wherein said compound isoleocanthal.
 18. The method of claim 17 wherein said oleocanthal is the(−)-enantiomer.
 19. The method of claim 17 wherein said oleocanthal isthe (+)-enantiomer.
 20. An animal repellent comprising an effectiveamount of a compound of the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅.
 21. The animal repellent of claim 20 wherein said compound isoleocanthal.
 22. The animal repellent of claim 21 wherein saidoleocanthal is the (−)-enantiomer.
 23. The animal repellent of claim 21wherein said oleocanthal is the (+)-enantiomer.
 24. The animal repellentof claim 20 wherein said repellent is in the form of a propellent spray.25. A method for treating a sore throat comprising administering to apatient with a sore throat an effective amount of a compositioncomprising a compound of the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅; and a pharmaceutically acceptable carrier.
 26. The method ofclaim 25 wherein said compound is oleocanthal.
 27. The method of claim26 wherein said oleocanthal is the (−)-enantiomer.
 28. The method ofclaim 26 wherein said oleocanthal is the (+)-enantiomer.
 29. The methodof claim 25 wherein said composition is in the form of a lozenge. 30.The method of claim 25 wherein said composition is in the form of aspray.
 31. A method of preserving food comprising contacting a food withan effective amount of a compound of the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅.
 32. The method of claim 31 wherein said compound isoleocanthal.
 33. The method of claim 32 wherein said oleocanthal is the(−)-enantiomer.
 34. The method of claim 32 wherein said oleocanthal isthe (+)-enantiomer.
 35. The method of claim 31 wherein said compound isincorporated into a film.
 36. The method of claim 31 wherein saidcompound is coated onto a packaging material which is in contact withsaid food.
 37. The method of claim 31 wherein said compound is addeddirectly to said food.
 38. A method of repelling animals from an ediblesource comprising adding to an edible source an effective amount of acompound comprising the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅.
 39. The method of claim 38 wherein said compound isoleocanthal.
 40. The method of claim 39 wherein said oleocanthal is the(−)-enantiomer.
 41. The method of claim 39 wherein said oleocanthal isthe (+)-enantiomer.
 42. The method of claim 38 wherein said compound isadded to an edible source otherwise susceptible to consumption by birds.43. The method of claim 38 wherein said compound is added to an ediblesource otherwise susceptible to consumption by non-human mammals. 44.The method of claim 38 wherein said compound is added to an ediblesource which is toxic to animals upon consumption.
 45. The method ofclaim 38 wherein said edible source is antifreeze.
 46. A method ofinhibiting sweetness perception in an edible source comprising adding toan edible source a sweetness inhibiting amount of a compound of theformula:

wherein: R₁ and are independently H or OR₅ R₂ and R₃ are independentlyCHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X is O, NH or CH₂ Yis C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, or CH—COOR₅. 47.The method of claim 46 wherein said compound is oleocanthal.
 48. Themethod of claim 47 wherein said oleocanthal is the (−)-enantiomer. 49.The method of claim 47 wherein said oleocanthal is the (+)-enantiomer.50. A method of treating a cold comprising administering to a patient inneed of treatment a composition comprising an effective amount of acompound of the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅.
 51. The method of claim 50 wherein said compound isoleocanthal.
 52. The method of claim 51 wherein said oleocanthal is the(−)-enantiomer.
 53. The method of claim 51 wherein said oleocanthal isthe (+)-enantiomer.
 54. The method of claim 50 wherein said compound isadministered as a nasal lavage or mist.
 55. A method of treating apatient with an inflammatory disorder comprising administering to apatient with an inflammatory disorder an effective amount of acomposition comprising a compound of the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅; wherein said composition alleviates inflammation in saidpatient.
 56. The method of claim 55 wherein said compound isoleocanthal.
 57. The method of claim 56 wherein said oleocanthal is the(−)-enantiomer.
 58. The method of claim 56 wherein said oleocanthal isthe (+)-enantiomer.
 59. (canceled)
 60. The method of claim 55 whereinsaid inflammatory disorder is selected from the group consisting ofpsoriasis, cancer, asthma, allergic rhinitis, respiratory distresssyndrome, inflammatory bowel disease, Chron's disease, gastritis,irritable bowel syndrome, ulcerative colitis, migraine, periarteritisnodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma,type I diabetes, myasthenia gravis, multiple sclerosis, sorcoidosis,ischemic kidney disease, nephrotic syndrome, Bechet's syndrome,polymyositis, gingivitis, conjunctivitis, vascular disease myocardialischemia, heart disease, and stroke.
 61. A method of inhibiting growthof microorganisms comprising contacting microorganisms with an effectiveamount of a composition comprising a compound of the formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅; wherein said composition inhibits the growth ofmicroorganisms.
 62. The method of claim 61 wherein said compound isoleocanthal.
 63. The method of claim 62 wherein said oleocanthal is the(−)-enantiomer.
 64. The method of claim 62 wherein said oleocanthal isthe (+)-enantiomer.
 65. The method of claim 61 wherein said compositionis incorporated into sutures or bandages.
 66. The method of claim 61wherein said composition is applied to a wound.
 67. A method forscreening for genes associated with oleocanthal sensitivity comprisingcontacting an expression library formed from an oleocanthal-responsivecell with oleocanthal conjugated to a detectable label; identifying anexpression clone that binds said oleocanthal; and sequencing thepolynucleotide that encodes the expression clone, thereby identifying agene associated with oleocanthal sensitivity.
 68. A method of screeningfor candidate genes associated with oleocanthal sensitivity comprisingidentifying differentially expressed genes in oleocanthal-responsivecells, obtaining sequences of said differentially expressed genes,comparing sequences of said differentially expressed genes andcorrelating similarity of said sequences to known that of tastereceptors, wherein high homology to a taste receptor thereby identifiesa candidate gene associated with oleocanthal sensitivity.
 69. A purified(−) isomer of the formula:


70. A purified (+) isomer of the formula:


71. A method of treating pain in a patient comprising administering to apatient an effective amount of a composition comprising a compound ofthe formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅; wherein said composition alleviates inflammation in saidpatient.
 72. The method of claim 71 wherein said compound isoleocanthal.
 73. The method of claim 72 wherein said oleocanthal is the(−)-enantiomer.
 74. The method of claim 72 wherein said oleocanthal isthe (+)-enantiomer.
 75. A method of purifying oleocanthal comprisingextracting olive oil with 80:20 volume/volume methanol:water to obtain aphenolic extract; pre-fractionating said phenolic extract on a C18 solidphase extraction cartridge; separating oleocanthal by reversed-phaseHPLC in a weak gradient and selecting a fraction containing throatirritating activity, thereby purifying oleocanthal.
 76. A method ofpreventing a neurodegenerative disorder comprising administering to apatient an effective amount of a composition comprising a compound ofthe formula:

wherein: R₁ and R₄ are independently H or OR₅ R₂ and R₃ areindependently CHO, or COOR₅ R₅ is a H, C₁-C₅ alkyl, or a glycoside X isO, NH or CH₂ Y is C═CHCH₃, or CH—COOR₅ Z is C═O or CH—OR₅ A is CH₂, orCH—COOR₅.
 77. The method of claim 76 wherein said compound isoleocanthal.
 78. The method of claim 77 wherein said oleocanthal is the(−)-enantiomer.
 79. The method of claim 77 wherein said oleocanthal isthe (+)-enantiomer.
 80. The method of claim 76 wherein said compositionprevents production of Aβ42 in said patient.
 81. The method of claim 76wherein said neurodegenerative disorder is selected from the groupconsisting of Alzheimer's disease and cognitive impairment.